The long-term objectives of this proposal are to understand the fundamental mechanisms by which human hepatitis B virus (HBV) X gene regulates gene expression, alters cellular growth and proliferation and influences the production of hepatocellular carcinoma (HCC). The correlation between chronic HBV infection and development of HCC is now well established, but an understanding of the mechanisms which regulate susceptibility to chronic viral infection and replication, and the basis for carcinogenesis are only beginning to emerge. The regulatory activity of the HBV X gene product, a transcriptional activating (transactivating) protein which stimulates promoters transcribed by both RNA polymerase II and III, implicates it in control of many critical aspects of the viral life-cycle. Experiments have been outlined to fully characterize the multiple transcriptional activities of the X protein. A set of X protein mutants have been constructed and will be used to identify structural elements and additional polypeptides encoded by the X gene required for stimulation of both classes of promoters. Because the X protein activates a diverse group of promoters and is predominantly located in the cell cytoplasm, it very likely functions by interacting with multiple cellular polypeptides involved in gene regulation. To identify these cellular factors, antibodies have been prepared against the X protein, and X expressing cell-lines and recombinant adenovirus vectors have been developed. Experiments are outlined to utilize these reagents to identify interacting cellular proteins. These studies should provide basic insights into the regulatory mechanisms of HBV during replication and cellular transformation. Little is known about the influence of HBV gene expression on cellular growth and proliferation. Results from this lab demonstrate that hepatocytes transformed with the HBV X gene display profound alterations in growth. A number of transactivating proteins possess the ability to influence cell growth and differentiation, possibly encoded by activities distinct from transcriptional stimulation. Studies have therefore been proposed to more fully explore the ability of the X gene to alter hepatocyte growth and differentiation, and to investigate the biological significance of this activity in HBV replication and contribution to the development of HCC. The specific aims of this proposal are: (1) To characterize the multiple functional activities of the HBV X protein and to determine whether they can be located to different structural elements or polypeptides encoded by the X gene. (2) To identify cellular polypeptides that likely interact with the X protein, some of which could be fundamental mediators of transcriptional regulation and cell growth. (3) To investigate the influence of the X protein on cell growth and proliferation. (4) To determine the potential role of the X protein in HBV replication.